1. Field of the Invention
This application relates generally to analytical devices such as liquid chromatography systems and, more particularly, to an inline flow rate meter with auxiliary fluid injection and detection for measuring volumetric flow rates.
2. Description of the Related Art
Chromatography is used for separating a sample into its various components. In more detail, chromatography is a group of analytical methods for taking a sample (e.g. a complex mixture) and separating its component substances, or analytes, from one another. In general, analytical chromatography is used to determine the existence and sometimes the concentration of analytes in a sample.
All chromatography systems involve two phases (states) of matter; a stationary phase and a mobile phase that carries the mixture through or past the stationary phase. The stationary phase is a solid and the mobile phase is a fluid, i.e. a liquid or a gas. As the mobile phase containing the mixture moves through the stationary phase, the components separate from one another because the components have different affinities for the two phases and thus move through the system at different rates. A component with a high affinity for the mobile phase moves relatively quickly through the chromatographic system, whereas one with a high affinity for the solid phase moves more slowly. The fast moving components, therefore, will be separated from the slow moving components.
Paper Chromatography
Paper chromatography is a simple and, to the layman, most familiar form of chromatography. It is very visual in nature. In paper chromatography, the stationary phase is usually a strip of porous paper, i.e. filter paper. A small dot or line of the mixture to be separated is placed on the paper strip, near its lower end, and then the paper strip is dipped into a liquid solvent (the mobile phase) that travels up the paper through capillary action—like a wick. The mobile phase reaches the mixture and then carries the mixture's different components along the paper. How fast each component moves along the paper depends on its relative affinity for the mobile and stationary phases. The components with the weakest attraction for the paper or greater affinity for the mobile phase travel faster than those that cling to the paper. For example, if the mobile phase is more polar than the stationary phase, the more polar components of the mixture will tend to move more quickly than the less polar components. The resulting separation visually shows the mixture's separate components.
Column-Based Liquid Chromatography
Column-based Liquid chromatography, usually just called liquid chromatography or LC, is a more complex method of chromatography where the stationary phase is contained within a so-called “column” rather than consisting of a sheet of paper. The mobile phase is usually a liquid solvent of one kind or other and the mixture to be analyzed is dissolved in or carried by the solvent.
Classic liquid chromatography is based on gravity. In such case, the column is a vertical tube that has been packed with a bed of suitable powder (the stationary phase) and gravity is the force that moves the mobile phase through the column. The components of the mixture that are carried through the column by the mobile phase can be visually identified if the column is clear and the components are differently colored, or they can be analyzed based on suitable measurements and the time that each component takes to travel through and exit the column.
Liquid chromatography often uses a pump in lieu of gravity. In a pump-based system, a primary pump is used to force the mobile phase through the column. In such case, the column may actually be horizontal. As the mobile phase exits the far end of the column, it transports the mixture's components out of the column, one after the other, and into a detector of one kind or another for compositional analysis based on measurement and the time that the component took to travel through and exit the column.
High Performance Liquid Chromatography (HPLC)
High Performance Liquid Chromatography (HPLC), sometimes aptly called high-pressure liquid chromatography, is another type of column-based chromatography that, like all forms of chromatography, uses a mobile phase to move the mixture through a stationary phase. HPLC provides high resolution results in a comparatively short period of time by using a relatively high pressure pump to force the solvent (the mobile phase) through relatively small diameter columns (e.g. 4.6 mm) that are packed with a stationary phase of very small particles (e.g. 1.5, 3, 5, or 10 μm, or microns) having a narrow particle size distribution (e.g. within 10% of the mean).
In order to achieve a separation using HPLC, we need to create a flow of mobile phase through the column. To do this, tubing is connected to the end of the column. Because of the small size of the particles in the column, the fluid needs to be pushed through the column at high pressure to get a reasonable flow rate.
In HPLC, as with traditional liquid chromatography, the stationary phase consists of a column (i.e. a metal tube) that has been packed with a solid (e.g. silica particles) and the unknown mixture to be analyzed is mixed with a mobile phase that consists of a suitable solvent. In HPLC, the liquid forming the mobile phase is forced through the column with high pressure pumps. If one regards the high pressure pump as a primary pump, then its liquid output is a primary fluid.
The solvent used is a matter of choice. HPLC usually involves a mixture of solvents that are chosen to provide the correct amount of polarity for a given separation. For example, the mixture could be comprised of 60% acetonitrile or ACN (less polar) and 40% water (very polar).
The typical HPLC pump produces a very high pressure, e.g. 15,000 Kilopascals (˜2,175 PSI) or 150× that of the atmosphere (standard atmospheric pressure is about 101.325 kPA). In HPLC, if a single sample is to be analyzed, a hypodermic syringe is usually used to inject the sample into the solvent stream via an injection port. Alternatively, and given the appropriate equipment, the operator can analyze several samples that are pre-injected into a plurality of vials that are then placed in a so-called autosampler that will run them in order without human intervention.
FIG. 1 is a simplified diagram of an exemplary HPLC system 10. As shown, the HPLC system 10 comprises a reservoir 20 containing a liquid solvent 21, a primary pump 30 that produces a primary fluid flow within a conduit that begins with tube 32, a sample vial containing a sample 41, an injector 50, a separation column 60, a detector 70, and a waste reservoir 80. In this particular system 10, the detector 70 is configured to display the chromatogram on a computer 71. In the drawing, the output paths of the injector 50, column 60, and detector 70 are identified with reference numbers 52, 62, and 72, respectively. In general, a conduit for transporting the fluid through the analytical instrument comprises the combined flow path through segments 32, 52, 62, and 72.
FIG. 2 illustrates an exemplary primary pump 30. The illustrated pump 30 uses a reciprocating piston that is driven by a motor, and a pair of check valves, but the pump 30 can be of any suitable construction. The pump 30, however, must be able to maintain accurate flow rates for accurate analysis in the HPLC system 10.
In operation, the pump 30 draws solvent 21 from the reservoir 20 via tube 22 and outputs a high pressure, primary fluid flow via tube 32. The injector 50 functions to place the sample 41 into the high pressure flow 32 so that the sample is transported to the column 60 as a homogenous, low-volume plug within tube 52. The pump 30 continues to force the solvent 21 and sample 41 into one end of the column 60 and, ultimately, the solvent 21 and separated components of the sample 41 emerge from the other end of the column 60 at which point the separated components of the sample 41 are analyzed by a suitable detector 70 that generates a chromatogram that is indicative of the components present. One common type of detector uses UV light. Another type of detector measures refractive index. There are many different kinds. Eventually, the solvent 21 and components of the sample 41 are dispensed into the waste reservoir 80.
The time that it takes a component to travel from the injection port 50, through the column 60, and then reach the detector 70 is called its “retention time” and it can be used to help identify the components and overall mixture 41. The HPLC system 10 typically produces a chromatograph that shows peaks of some characteristic response being measured by the detector 70, the peaks being horizontally spread out along the abscissa (X-axis) as a function of retention time.
The retention time is a function of the component's interaction with the stationary phase, the composition of the solvent, and of special significance to this invention, the flow rate of the mobile phase or primary fluid flow. In order to produce an accurate HPLC chromatograph based on retention time, the flow rate must be accurately known and maintained.
Existing Flow Rate Measurement Techniques
The pump used in an HPLC system must consistently provide accurate, stable, and known flow rates. In certain situations, the operator can directly verify the flow rates for different flow rate settings through actual time and volume measurements. This is done by diverting the flow of solvent and by measuring the amount of time required to collect a known amount of solvent. The solvent can be diverted for measurement on the upstream or downstream side of the column. In either case, the user temporarily diverts the flow of solvent into a volumetric measuring device (e.g. a volumetric flask, a graduated cylinder, or a pipette) and uses a timer to measure how much fluid flows into the measuring device in a given amount of time. For example, if one is trying to verify the flow-rate accuracy at 2 mL/min, one could use a stopwatch to measure the time that it takes to collect 25 mL of effluent that is diverted into a 25 mL volumetric flask. At exactly 2 mL/min, it would take 12.5 minutes to fill the flask. For reasonable operation, one would expect to have a flow rate accuracy of +/−1% of the set flow rate.
Diverted flow rate measurements detrimentally take the system offline and often take more time than desired.
Moreover, diverted flow rate measurements are only suitable for relatively high flow rates. The lowest flow rate that can reasonably be measured and verified with a direct measurement technique is about 1 ml/minute. However, certain HPLC applications often require verification of very low flow rates that are a few orders magnitude lower.
Another technique for measuring flow rate is based on temperature as disclosed, for example, in U.S. Pat. No. 6,813,944 assigned to Sensiron AG. The device disclosed in the 944 patent includes a heat source and two temperature sensors that correlate the flow rate with a measured temperature gradient. In essence, it is a parametric measurement of flow rate, and not a direct measurement.
There remains a need, therefore, for an improved flow meter that operates quickly, does not require diversion, does not interrupt the analytical process, directly measures the flow rate, and can be effectively used with very low flow rates.